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Sample GSM3188790 Query DataSets for GSM3188790
Status Public on Jun 01, 2020
Title week6_DN3_3
Sample type SRA
Source name library of 96 WT DN3 cells
Organism Mus musculus
Characteristics strain background: C57BL/6
genotype/variation: wild type
Sex: female
age/developemental stage: 5-6 weeks
tissue: Thymus
cell type: DN3 cells
Treatment protocol Maf KO, Sox13 Ko and Rorc KO cells were kept frozen in 90 % fetal bovine serum (FBS) and 10 % Dimethyl sulfoxide (DMSO) -80ºC before performing antibody staining, FACS sorting and single cell RNA sequencing.
Growth protocol WT mice were kept in the animal facility of the Max Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany. Maf KO mice were kept in the animal facility of The Kimmel Center for Biology and Medicine of the Skirball Institute, New York University School of Medicine, New York, USA. Sox13 KO mice were kept in the animal facility of Institut für Biochemie, Emil-Fischer-Zentrum, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany. Rorc KO mice were kept in the animal facility of Institute of Medical Microbiology and Hygiene, University Medical Center Freiburg, Freiburg, Germany.
Extracted molecule total RNA
Extraction protocol Thymocytes were isolated using mechanical dissociation of thymus. Since double negative (DN) thymocytes are rare, we performed the magnetic enrichment for DN thymocytes.
As described in mCEL-Seq2 protocol (Hashimshony et al. 2016 and Herman et al. 2018)
Adapted from TruSeq Small RNA Library Preparation Protocol
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 2500
Description library of 96 WT DN3 cells
Data processing For image aquisition, intensity extraction and basecalling HiSeq Control Software 2.0.2, RTA 2.4.11 / Recipe Fragment​ was used. Conversion of bcl2fastq files was performed using bcl2fastq
Paired end reads were aligned to the transcriptome using bwa (version 0.6.2-r126) with default parameters. The transcriptome contained all gene models based on the mouse ENCODE VM9 release downloaded from the UCSC genome browser comprising 57,207 isoforms derived from 57,207 gene loci with 57,114 isoforms mapping to fully annotated chromosomes (1 to 19, X, Y, M). All isoforms of the same gene were merged to a single gene locus. Furthermore, gene loci overlapping by >75% were merged to larger gene groups. This procedure resulted in 34,111 gene groups.
The right mate of each read pair was mapped to the ensemble of all gene loci and to the set of 92 ERCC spike-ins in sense direction. Reads mapping to multiple loci were discarded. The left read contains the barcode information: the first six bases correspond to the cell specific barcode followed by six bases representing the unique molecular identifier (UMI). The remainder of the left read contains a poly(T) stretch. The left read was not used for quantification. For each cell barcode, the number of UMIs per transcript was counted and aggregated across all transcripts derived from the same gene locus.
Based on binomial statistics, the number of observed UMIs was converted into transcript counts (Gruen et al., 2014).
Genome_build: ENCODE VM9
Supplementary_files_format_and_content: CSV files, columns represent each cell barcode (total barcodes used = 192), rows represent the geneid and the values in the file are the quantified number of transcripts.
Submission date Jun 13, 2018
Last update date Jun 01, 2020
Contact name Sagar -
Organization name University Medical Center Freiburg
Department Department of Internal Medicine II
Lab Sagar
Street address Hugstetter Straße 55
City Freiburg
ZIP/Postal code 79106
Country Germany
Platform ID GPL17021
Series (1)
GSE115765 Deciphering the regulatory landscape of fetal and adult gd T-cell development at single-cell resolution
BioSample SAMN09407135
SRA SRX4211867

Supplementary file Size Download File type/resource
GSM3188790_week6_DN3_3.coutt.csv.gz 432.9 Kb (ftp)(http) CSV
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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